Isoliensinine ameliorates cognitive dysfunction in AlCl3/D-gal-induced Alzheimer's disease-like mice by inhibiting the calcium signaling pathway

Abstract

Ethnopharmacological relevance: The embryos of lotus (Nelumbo nucifera Gaertn.) is a famous traditional Chinese medicine used to treat insomnia, memory decline, and dementia for a long time. However, the underlying material basis and mechanisms of this medicine are still unclear. Isoliensinine (IL) is a major alkaloid derived from lotus embryos. Our previous research has demonstrated that IL can exert strong anti-inflammatory and neuroprotective effects in vitro.

Aim of the study: To reveal the underlying therapeutic effect and mechanism of IL on Alzheimer's disease (AD)-like mice induced by AlCl₃ and D-galactose (D-gal) in vivo.

Materials and methods: The AD-like mice were modeled by intragastric injection (i.g.) of AlCl₃ (20 mg/kg/day) and intraperitoneal injection (i.p.) of D-gal (120 mg/kg/day) for 8 weeks. Starting from the third week, AD-like mice were treated with IL (1, 3, or 10 mg/kg/day; i.p.) for 6 weeks. Cognitive impairment in AD-like mice was evaluated through some behavioral experiments including nest building, open field, novel object recognition, Y maze, and Morris water maze tests. The cortex and hippocampus (DG, CA1, and CA3) regions were analyzed as follows: Neuronal pathological changes and neurofibrillary tangles (NFTs) formation were observed by hematoxylin-eosin (HE) and silver staining, respectively; The production of Aβ plaques and the activation of microglia and astrocytes were detected by immunohistochemistry; The levels of Ca²⁺ levels were determined by the ortho-cresolphtalein complexone method. The levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) were analyzed using the ELISA kits. The expression of CaM, p-CaMKII, Calpain, CDK5, p35/p25, p-Tau, ADAM10, BACE1, PSEN1, APP, Aβ_1-42, p-IκBα, and IκBα were evaluated by western blotting.

Results: IL (1, 3, and 10 mg/kg) treatment effectively ameliorated cognitive impairment in AD-like model mice. IL inhibited the decrease of brain index and body weight in AD-like mice and alleviated neuronal damage in the cortex and hippocampus (DG, CA1, and CA3). IL decreased the levels of Ca²⁺ and reduce high expression of CaM and Calpain in the cortex and hippocampus of AD-like mice. IL treatment did not affect the expression of CDK5 but inhibited the expression of p-CaMKII and p25/p35, and reduced Tau phosphorylation and NFTs formation. IL also down-regulated the high expression of Aβ_1-42 and APP and regulated the expression of APP-cleavage secretase (reducing the expression of BACE1 and PSEN1, while increasing the expression of ADAM10), thereby inhibited the production of Aβ plaques in AD-like mouse brain. Moreover, IL inhibited the phosphorylation and degradation of IκBα, as well as the production of inflammatory cytokines (TNF-α, IL-6, and IL-1β), and prevented the activation of microglia and astrocytes in AD-like mice.

Conclusions: IL has a significant therapeutic effect on pathological alterations and cognitive impairment in AlCl₃ and D-gal-induced AD-like mice, indicating that IL may have the potential to treat AD. The anti-AD activity of IL may be associated with its regulation of the Ca²⁺ homeostasis and downstream signaling molecules such as CaM and Calpain.

Keywords: AlCl(3); Alzheimer’s disease; Calcium signaling pathway; D-gal; Isoliensinine.